1. Field of the Invention
The present invention relates to a novel method for immobilizing polypeptides of interest and to an immobilized polypeptide. In particular, the invention provides a method for immobilizing fusion polypeptides or proteins to surfaces by fusing said polypeptides to adhesion polypeptides or proteins which have the capability of binding to the surface.
2. Description of Related Art
Protein immobilization is important for many diagnostic and biosensor applications and in, for example, immobilized enzyme reactors. The quality and properties of the adsorbed layer is often of importance for the performance of the application. Often immobilization is achieved by chemical cross-linking with glutaraldehyde (or a similar reagent), spontaneous adsorption (physical adsorption), or by coupling to reactive groups on a surface (for example amine-reactive groups). The drawback of these methods is often that they are difficult to control and may involve loss of activity of the immobilized protein. In physical adsorption loss of activity is due to denaturation of the target protein and when using cross-linking or coupling it is due to new chemical bonds that either restrict the accessibility of the protein or alter the chemical properties of the target protein. Additionally, these immobilization methods do not offer a general way of orienting the target protein in any desired fashion.
Protein monolayer formation has been described using the Langmuir-Blodgett technique, in which molecules are mechanically forced together by compression on the surface of water using a special apparatus, and then transferred onto a support. For some proteins, layers can be manufactured either directly in this manner or, for example, first making a biotinylated lipid layer, to which an avidin layer is bound and to which in turn a biotinylated target protein can be bound. The problem involved in this technique is that most proteins do not form layers at the interphase which can be transferred, and that special equipment is needed.
It is of interest that a rapid and easy immobilization method is developed. Preferred properties of an immobilization method would, for example, be that there is no need for special equipment or that there is no need for formation of monolayers. A very important property of an immobilization method is that the protein of interest is not denatured. It is of advantage also that in the method there are no chemical cross linking steps, because that would increase the probability of denaturation of the protein.
Preferred properties would furthermore be that the protein of interest can be oriented in a desired fashion to the solid phase, that the production of the protein to be immobilized is easy and that the protein surface layer is durable. It is also of practical value that layers which have a certain density of the polypeptide of interest, can be made. It can also be of value that the immobilized protein can be easily removed from the solid phase under certain conditions.
Fusion polypeptides attached to a solid carrier are known from WO 8803563, WO 0179559, U.S. Pat. No. 6,210,929, WO 0134902, WO 9325533, U.S. Pat. No. 5,849,506, from Regimbald et al. (1996) and Janelli et al. (1997). WO 0058342 describes a fusion polypeptide, the other party of which is an adhesion polypeptide.
It is known that proteins used as antigens in enzyme-linked immunosorbent assays (ELISA) or in similar assays where a polypeptide is bound to a surface, can lose or acquire changed antigen binding properties or other properties, and thus cause problems in the assay. In one way to avoid this, the antigen needs to be biotinylated and then bound to a surface which previously has been coated with avidin or streptavidin in an extra step.
Wessels (1997) has suggested that hydrophobins may be used as an intermediate layer to attach cells, proteins, such as antibodies, and small ligands to hydrophobic surfaces. SC3 hydrophobin has been shown to coat a hydrophobic gold surface. It was suggested that at the exposed hydrophilic side of surface-bound SC3 film, mannose residues can be oxidized with periodic acid without disturbing the binding to the gold, while the generated aldehyde groups were suggested to be coupled to amino groups of a protein by a Schiff based reaction. This approach has the disadvantage that new chemical bonds are formed that may either restrict the accessibility of the protein or alter the chemical properties of the target protein.